The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII.
In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.
Bom
V J
VJ
Haemostasis and Thrombosis Research Unit, University Hospital Leiden, The Netherlands.
Bertina
R M
RM
D001059:Apoproteins / Q000502:physiology*; D002122:Calcium Chloride / Q000494:pharmacology*; D004789:Enzyme Activation; D015942:Factor VIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D013925:Thromboplastin / Q000502:physiology*
D001059:Apoproteins; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa; D002122:Calcium Chloride